Phenolic Composition and Antioxidant Properties of Some Spices
Aqueous methanolic extracts of 9 spices were investigated for their phenolic compounds composition and antioxidant properties. The spices investigated were, Laurel noblis (bay leaves), Rosimarinus officinalis (rosemary), Salvia officinalis (sage), Origanum marjorana (marjoram), Origanum valgare (oregano), Cinnamonum zeylanicum (cinnamon), Petroselium crispum (parsley), Ocium basilicum (sweet basil) and Mentha peperita (mint). The phenolic compound contents were determined by the Folin Ciocalteu, tannin binding assay and High Performance Liquid Chromatography (HPLC). The antioxidant properties were determined by the reducing power assay, radical scavenging assay and the β-carotene linoleic acid model system. Oregano had the highest total phenolic compound concentration of 15.83 mg GAE g-1 and cinnamon had the highest polyphenolic compound concentration of 13.66 mg GAE g-1. Marjoram had the highest proportion of simple phenolic compounds of 95.57%. Ascorbic acid was used as a control in all the antioxidant assays. At 25 mg mL-1 cinnamon and oregano recorded a high reducing power activity with absorbance of 0.12, while parsley had the lowest activity with absorbance of 0.075 at 655 nm. Cinnamon and marjoram had the highest radical scavenging activities of 92.0 and 91.3% respectively while at a concentration of 5 mg mL-1, parsley had the least radical scavenging activity of 47.90%. Cinnamon and oregano had the highest antioxidant activities of 61.76 and 58.28%, respectively while sweet basil had the lowest activity of 6.67%. Most of the spices showed better antioxidant properties than ascorbic acid. HPLC analysis detected gallic acid, protochatechuic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic acid, caffeic acid, p-coumaric acid and ferullic acid in the studied spices.
Phenolic compounds are secondary plant metabolites that possess in common an aromatic ring bearing one or more hydroxyl substituents. Phenolic compounds are water soluble and may occur combined with a sugar molecule, as glycosides (Harbone, 1998). Phenolic compounds are divided into sub-groups and these include, phenols, phenolic acids, phenylpropanoids, flavonoids, flavones, glycoflavonones and biflavonyls, minor flavonoids, aurones, flavonones, dihydrochalcones, isoflavones, xanthones and stilbenes, hydrolysable and condensed (proanthocyanidins) tannins and quinines (Harbone, 1998; Strack, 1997).
Phenolic compounds have diverse biological activities ranging from toxicity
to hormonal mimicry and act as cell wall material, colorful attractants
for birds and insects helping seed dispersal and pollination. Phenolic compounds
also act as defense mechanisms of plants under different environmental stress
conditions such as wounding, infection, excessive light or UV irradiation (Harbone,
1998). The biological potency of phenolic compounds includes possible pharmacological
value (Ingold, 1960). Phenolic compounds have long been recognized to possess
antiallergenic, anti-inflammatory, antiviral and antiproliferative activities.
Phenolic compounds have some antioxidant activity. They are able to terminate free radicals and chelate metal ions that are capable of catalyzing formation of oxygen reactive species that promote lipid peroxidation. Phenolic compounds interfere with oxidation of lipids and other free radicals by rapid donation of a hydrogen atom or electrons to the oxidized molecule or radicals. The resultant radical from the reaction of phenol with lipid radical is stabilized by the delocalisation of unpaired electrons around the aromatic ring (Ingold, 1960). Stability of the phenoxy radical reduces the rate of propagation of auto-oxidation chain reactions because the propagation reaction is slow and the bulky groups of 2 and 6 positions offers steric hindrances in the region of the radical and reduces the rate of propagation (Hudson, 1990). Phenolic compounds are understood to induce the cellular antioxidant system; quercetin and flavonoids were found to increase the intra cellular concentration of glutathione by approximately 50%. Flavonoids are important in the modulation of γ-glutamylcysteine synthase in both cellular antioxidant defenses and detoxification of xenobiotics. Glutathione is important in redox regulation of transcription factors and enzymes for signal transduction. It is therefore likely that polyphenols mediated regulation of glutathione alters cellular processes (Hudson, 1990; Yoshida et al., 1997).
Spices are plant products added to food to contribute towards aroma, test, flavor, color and pungency. These attributes are believed to be due to the presence of phenolic compounds in spices. Spices are derived from the bark, flowers, leaves, rhizomes, roots, seeds, or the entire plant organs. The use of spices is widely spread in Asian countries. Historically spices were exploited for their anti-microbial properties to preserve meat products.
There are no reports on the antioxidant capacities of Laurel noblis (bay leaves), Rosimarinus officinalis (rosemary), Salvia officinalis (sage), Origanum marjorana (marjoram), Origanum valgare (oregano), Cinnamonum zeylanicum (cinnamon), Petroselium crispum (parsley), Ocium basilicum (sweet basil) and Mentha peperita (mint) cultivated in Zimbabwe. The aim of the study was to determine the amount of phenolic compounds content and antioxidant activities of the selected spices. All the studied spices are native in the Mediterranean and cool climates but they can all be grown in gardens in other climates. In Zimbabwe they are grown at a small scale in isolated farms in the eastern highveld and the watershed regions.
MATERIALS AND METHODS
The chemical standards used were all of analytical grade. Folin-Ciocalteau,
gallic acid, catechin, vanillic acid, caffeic acid, p-coumaric acid, protocatechuic
acid, ferulic acid, p-hydroxy-benzoic acid, p-hydroxybenzaldehyde, Nitroblue
tetrazolium salt (NBT), 1, 1-diphenyl-2 picrylhydrazyl radical (DPPH•),
phenazine methosulphate (PMS), ascorbic acid, trichloroacetic acid (TCA) and
potassium ferricyanide were obtained from Sigma-Aldrich Chemie (Steinheim, Germany).
Reduced nicotinamide adenine dinucleotide (NADH) was obtained from Boehringer,
Manheim, Germany. Sodium carbonate, methanol (HPLC grade), ascorbic acid, HCl,
ethyl acetate, diethyl ether, anhydrous Na2SO4, acetonitrile
(super purity solvent), acetic acid and ferric ammonium sulphate were obtained
Folin-Ciocalteu reagent (1 N), 20% sodium carbonate, standard gallic acid (0.5 mg mL-1), 50% methanol in distilled water (1:1 v/v) and ferric reagent (2% ferric ammonium sulfate in 2M HCl) were used for analysis.
Procurement of Samples
All samples were obtained from the local supermarkets and they were all
processed in Zimbabwe. The spices were packaged by local companies namely New
Seasons, Mr Tasties Foods and Spice Foods. The spices obtained were analyzed
before the expiry date. They were manufactured for food flavoring purposes.
Samples were kept at room temperature before extraction. The spices investigated
included the Libiatae family, Rosimarinus officinalis (rosemary),
Ocium basilicum (sweet basil), Salvia officinalis (sage), Origanum
valgare (oregano), Origanum marjorana (marjoram) and Mentha
peperita (mint), the Laurel family, Laurel nobilis (bay leaves)
and Cinnamonum zeylanicum (cinnamon) and the Umbellifereae family,
Petroselium crispum (parsley).
Extraction of Phenolic Compounds
Total phenolic compounds were extracted from the ground material as described
by Makkar (1999). Phenolic acids for HPLC analysis were extracted following
the method described by Pena-Neira et al. (2000).
Quantification of Phenolic Compounds
Total phenolic compounds were determined following the method by Makkar
(1999). Amount of tannins were determined using the method of Makkar and Goodchild
Antioxidant Activity Assays
The DPPH radical scavenging activity and the reducing power effects were
determined following the method by Kuda et al. (2005). The inhibition
of lipid oxidation was assayed using the method of Amin and Tan (2002).
HPLC Analysis for Phenolic Acids
Detection of phenolic acids was carried out by measuring absorbance at 280
nm according to Pena-Neira et al. (2000). After each run, the system
was reconditioned for 15 min before analysis of the next sample.
RESULTS AND DISCUSSION
Total Phenolic Compounds Determination
The concentration of the phenolic compounds in the spices ranged from 6.90
to 15.83 mg GAE g-1 and they were in the order oregano>cinnamon>sweet
basil>bay leaves>mint> sage>rosemary>parsley>marjoram (Fig.
1). The results are comparable to those obtained by Miliauskas et al.
(2003), who studied some culinary plants and obtained ranges of 4.30 to 37.90
mg GAE g-1. Wagensteen et al. (2004) obtained 19 mg GAE g-1
total phenolic compounds in some coriander plants. Ismail et al. (2004)
detected ranges of 11.07 to 71.67 mg GAE g-1 in selected vegetables.
Capecka et al. (2003) detected between 11.07 and 14.06 mg GAE g-1
total phenolic compound contents in some herbs. Variation of phenolic compounds
content arises due to several factors, which include the area of cultivation
and other environmental stresses (Makkar, 1999).
The Tannin-Binding Assay
Most phenolic compounds in plants occur as polyphenolic compounds (Fig.
2). Polyvinyl-polypyrolidone (PVPP) binds efficiently to tannins (Makkar,
1999). The concentration of tannins after treating the sample with (PVPP) was
found to range from 0.31 to 13.66 mg GAE g-1. The order of the concentration
of tannins in the studied spices was as follows: cinnamon>oregano>sweetbasil>mint>
bay leaves> parsley>sage>rosemary>marjoram.
The Reducing Power Assay
Increase in absorbance with increasing concentration indicated an increase
in reducing power. The phenolic compounds in methanolic extracts of spices were
able to reduce potassium ferricyanide to a ferrous state.
||The tannins concentration expressed as mg GAE g-1
of samples (a) and the simple phenolic compounds concentration expressed
as mg GAE g-1 sample (b)
Ascorbic acid is a known reducing agent. It can be deduced that phenolic compounds
in the spices are more potent reducing agents compared to ascorbic acid (Fig.
3). Ascorbic acid had an absorbance of 0.03 at 0.5 mg mL-1 and
at 25 mg mL-1 it had an absorbance of 0.04. For all the spices at
5 mg mL-1 the absorbencies ranged from 0.05 to 0.07 and at 25 mg
mL-1 the absorbencies ranged from 0.08 to 0.12. At 25 mg g-1
the reducing power of the spices investigated was decreasing as follows: cinnamon>oregano>marjoram>sage>rosemary>mint>sweet
basil>bayleaves>parsley. Marjoram exhibited high reducing power activity
though it had low levels of total phenolic compounds. The high reducing power
of marjoram may be due to its high levels of simple phenolic compounds. The
free phenols can easily donate their electrons to any electron-deficient substance.
The DPPH Radical Scavenging Assay
β-Carotene Linoleic Acid Model System
The radical scavenging activity of spices increases with increase in concentration
(Fig. 4). At 5 mg g-1 bay leaves had 91.1% radical
scavenging activity, rosemary 88.5%, marjoram 91%, sage 88.35%, oregano 89.8%,
cinnamon 92%, parsley 47.5%, sweet basil 90.1 and mint 83%. Most of the samples
managed to scavenge the DPPH radical to above 75% with the exception of parsley.
The results are comparable to those found by Mau et al. (2004) who
obtained 78.8, 79.4 and 94.1% DPPH scavenging activities from methanolic extracts
of Terminotomyces albuminosus, Grifola frondosa and Marchella
esculenta, respectively (Wagensteen et al., 2004).
The antioxidant activity
was measured by comparing the bleaching capacity
of sample with the control, which contained no antioxidant component. Cinnamon
and oregano had the highest activity with sweet basil having the least antioxidant
percentage activity of 6.67% (Table 1
||The reducing power activity of the studied spices
||The percentage scavenging activity on the free radical DPPH
of the nine spices
The activities of cinnamon and oregano are comparable to those obtained by
Ismail et al. (2004) from extracts of shallots, spinach, swamp cabbage,
cabbage and kale, which were 69.1, 66, 60.3, 59.3 and 50.2%, respectively. The
differences in the antioxidant activity of the spices could be due to the method
of cultivation used for the different spices and differences in environmental
factors they were exposed to, such as climatic growth conditions and duration
of storage. Characteristics of the phenolic compounds may affect the antioxidant
activity. Rice-Evans et al. (1995) suggests that the ortho-substitution
with electron donating alkyl or methoxy groups of flavonoids, increases the
stability of free radical and hence its antioxidant potential. The position
and degree of hydroxylation of phenolic compounds is of primary importance in
determining the antioxidant activity of phenolic compounds. The ortho and para
positions of hydroxyl groups contribute markedly to the antioxidant activity
while the meta position has little or no effect on the antioxidant.
HPLC was used to identify the phenolic compounds that were present in the
studied spices. The retention times of the standards were used to identify the
individual phenolic compounds that are found in the spices. Gallic acid, protochatechuic
acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic acid, caffeic acid,
p-coumaric acid and ferullic acid were positively identified in the studied
spices though they did not all occur in the same sample. Ferullic acid was common
in all the spices. Caffeic acid was found in all the spices expect in mint but
the peaks it produced were very small suggesting that it is found in very small
quantities in all the samples.
||The percentage antioxidant activity of the studied spices
showing the capacity of spices extracts to inhibit bleaching of β-carotene
||HPLC results showing the phenolic acid composition of the
Vanillic acid was found in all samples except in sweet basil. Only oregano
and sweet basil contained p-hydroxybenzoic acid and p-hydroxybenzaldehyde (Table
2). The HPLC results obtained are comparable to those we obtained in Ziziphus
mauritiana and Uapaca kiriana fruits (Muchuweti et al., 2005;
Muchuweti et al., 2006). The phenolic compounds identified are
widely found in plants. Schindler et al. (2005) detected p-hydroxybenzaldehyde,
p-coumaric acid, ferullic acid and vanillic acid in tomatoes.
The nine spices studied are important sources of potent phenolic compounds. Phenolic compounds from spices are valuable sources of antioxidants and they have shown to have generally better antioxidant properties than ascorbic acid. The spices exhibited strong antioxidant capacity in vitro and they may be potential sources of natural antioxidants.
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